Abstract: Chronic pain represents an immense clinical problem, with over 100 million Americans afflicted and an annual price tag exceeding half a trillion dollars, according to a recent report from the Institute of Medicine. Studies in our lab are designed to identify molecular, cellular, and circuit mechanisms of sensitization in pain pathways with the goal of identifying novel targets for analgesic intervention. Studies performed under the three previous terms of this grant identified a critical signaling cascade in central nucleus of the amygdala (CeA) neurons that underlies central pain sensitization. This pathway is initiated by metabotropic glutamate receptor subtype 5 (mGlu5) activation of extracellular signal-regulated kinase/ERK signaling, leading to increased firing of CeA neurons. This increase in excitability likely contributes to central sensitization associated with persistent pain. Our prior work, and that of several other groups, has demonstrated robust analgesic actions of mGlu5 antagonists in a variety of animal models of pain. In the previous round of funding of this award, our group subsequently found that mGlu5 antagonists also reduce pain in humans. We found that although mGlu5 activation is important at multiple points along the pain neuraxis, selective blockade of mGlu5 in the CeA or conditional knockout of mGlu5 in CeA neurons produces profound analgesia. These findings suggest that mGlu5 expressing neurons in the CeA represent a critical node of neuromodulation underlying the development of chronic pain. In the present application, we conduct a series of studies aimed at understanding circuit context of these neurons, identifying critical inputs, the type of plasticity that occurs at these synapses, and the major outputs of mGlu5-expresing CeA neurons. We will test whether CeA neurons activated in the context of pain sensitization are necessary and sufficient for the development of pain sensitization, ongoing pain and negative valence, and comorbid anxiety. In vivo 2-photon imaging and microendoscope cameras will be used to monitor activity of these neurons using the genetically-encoded Ca2+ sensor GCaMP6m, over days to weeks to determine how the properties of these neurons change during the transition from acute to persistent pain. We will ask whether the population of neurons responsive to heat, cold, or touch change over time, and whether altered activity of these neurons in persistent pain conditions can be normalized using treatments that reduce pain or comorbid anxiety. These studies employ a host of modern techniques including advanced viral tracing, genetic trapping, intravital calcium imaging, and optogenetic approaches, together with technologies developed in our lab for wireless optogenetic studies to address these important questions.